Catherine Frelin

AS602868, a pharmacological inhibitor of IKK2, reveals the apoptotic potential of TNF- α in Jurkat leukemic cells

NF-κB transcription factors promote survival in numerous cell types via induction of antiapoptotic genes. Pharmacological blockade of the IKK2 kinase with AS602868, a specific inhibitor that competes with ATP binding, prevented TNF-α-induced NF-κB activation in Jurkat leukemic T cells. While TNF-α by itself had no effect on Jurkat survival, the addition of AS602868 induced cell death, visualized by DNA fragmentation and sub-G1 analysis. A disruption of the mitochondrial potential followed by activation of caspases 9 and 3 was observed in cells treated by the combination TNF-α+AS602868. Quantitative real-time PCR demonstrated that AS602868 prevented TNF-α induction of the antiapoptotic genes coding for c-IAP-2, Bclx, Bfl-1/A1 and Traf-1. The use of a specific IKK2 inhibitor appears, therefore, as an interesting pharmaceutical strategy to increase the cells sensitivity towards apoptotic effectors.

Blocking NF-κB activation in Jurkat leukemic T cells converts the survival agent and tumor promoter PMA into an apoptotic effector

The transcription factor NF-κB promotes cell survival. Using a variant of Jurkat leukemic T cells expressing IκB-αΔN, a super-repressor of NF-κB activation we first show that the tumor promoter PMA could prevent Fas-induced apoptosis via activation of NF-κB. Moreover, we demonstrate that in the absence of NF-κB activation, PMA became a strong inducer of apoptosis through stimulation of the upstream caspases 8 and 9 as well as of the effector caspase 3. A RNase-protection analysis showed that PMA stimulated the expression of several known anti-apoptotic genes (TRAF1, TRAF4, c-IAP-1, c-IAP-2, Bfl-1, Bcl-xl). In the absence of NF-κB activation, these survival influences were strongly lowered revealing the apoptotic effect of PMA. These results suggest that NF-κB activation could be an important step in the tumor promoting effect of PMA.

NF‐κB inhibition triggers death of imatinib‐sensitive and imatinib‐resistant chronic myeloid leukemia cells including T315I Bcr‐Abl mutants

The Bcr‐Abl inhibitor imatinib is the current first‐line therapy for all newly diagnosed chronic myeloid leukemia (CML). Nevertheless, resistance to imatinib emerges as CML progresses to an acute deadly phase implying that physiopathologically relevant cellular targets should be validated to develop alternative therapeutic strategies. The NF‐κB transcription factor that exerts pro‐survival actions is found abnormally active in numerous hematologic malignancies. In the present study, using Bcr‐Abl‐transfected BaF murine cells, LAMA84 human CML cell line and primary CML, we show that NF‐κB is active downstream of Bcr‐Abl. Pharmacological blockade of NF‐κB by the IKK2 inhibitor AS602868 prevented survival of BaF cells expressing either wild‐type, M351T or T315I imatinib‐resistant mutant forms of Bcr‐Abl both in vitro and in vivo using a mouse xenograft model. AS602868 also affected the survival of LAMA84 cells and of an imatinib‐resistant variant. Importantly, the IKK2 inhibitor strongly decreased in vitro survival and ability to form hematopoietic colonies of primary imatinib resistant CML cells including T315I cells. Our data strongly support the targeting of NF‐κB as a promising new therapeutic opportunity for the treatment of imatinib resistant CML patients in particular in the case of T315I patients. The T315I mutation escapes all currently used Bcr‐Abl inhibitors and is likely to become a major clinical problem as it is associated with a poor clinical outcome.